U.S. patent number 4,557,931 [Application Number 06/446,231] was granted by the patent office on 1985-12-10 for antigenic compositions and methods for using same.
This patent grant is currently assigned to Regents of the University of California. Invention is credited to Leslie D. Cahan, Reiko F. Irie, Donald L. Morton, James C. Paulson, Tadashi Tai.
United States Patent |
4,557,931 |
Irie , et al. |
December 10, 1985 |
Antigenic compositions and methods for using same
Abstract
GM2 is a ganglioside present on the surface of tumors and
stimulates an appreciable immune response in mammals. It is useful
when coupled with a non-toxic protein carrier or mixed with an
adjuvent and injected parenterally of raising the anti-GM2 titer in
serum. GM2 is also valuable as a diagnostic agent.
Inventors: |
Irie; Reiko F. (Los Angeles,
CA), Tai; Tadashi (Los Angeles, CA), Morton; Donald
L. (Los Angeles, CA), Cahan; Leslie D. (Los Angeles,
CA), Paulson; James C. (Los Angeles, CA) |
Assignee: |
Regents of the University of
California (Berkeley, CA)
|
Family
ID: |
23771814 |
Appl.
No.: |
06/446,231 |
Filed: |
December 2, 1982 |
Current U.S.
Class: |
424/194.1;
435/7.23; 435/961; 424/184.1; 424/193.1; 424/450; 435/960; 530/403;
424/277.1 |
Current CPC
Class: |
G01N
33/57469 (20130101); A61K 9/1271 (20130101); G01N
33/574 (20130101); A61K 39/001171 (20180801); G01N
2474/20 (20210801); A61K 2039/6081 (20130101); Y10S
435/961 (20130101) |
Current International
Class: |
A61K
9/127 (20060101); A61K 39/00 (20060101); G01N
33/574 (20060101); A61K 039/00 (); G01N
033/54 () |
Field of
Search: |
;424/88 |
Other References
Tai et al.-Chem. Abst. vol. 99 (1983) pp. 137, 909s. .
Jonah et al.-Chem. Abst. vol. 89 (1978) pp, 104, 517c. .
Young et al.-Chem. Abst. vol. 92 (1980) p. 39574g. .
Uchida et al.-Chem. Abst. vol. 94 (1981) p. 26466x. .
Chem. Abst. Subj Index-10th Collect (1977-1981) pp. 23060cs and
26062cs..
|
Primary Examiner: Rosen; Sam
Attorney, Agent or Firm: Poms, Smith, Lande & Rose
Government Interests
This invention was made with Government Support under Grant Nos. CA
12582 and CA 30647 awarded by the Department of Health and Human
Services. The Government has certain rights in this invention.
Claims
We claim:
1. An antigenic conjugate which is immunoreactive with anti-GM2
antibodies produced by mamallian tumors said antigenic conjugate
consisting essentially of the oligosaccharide portion of GM2
conjugated to a non-toxic mammal compatible protein carrier.
2. An antigenic conjugate according to claim 1 wherein the
conjugated bond between said oligosaccharide and said non-toxic
protein carrier is formed by the glucose moiety of the
oligosaccharide.
3. An antigenic conjugate according to claim 1 wherein the
conjugated bond between said oligosaccharide and said non-toxic
protein carrier is formed by the amine group of the sphingosine
moiety of the ceramide portion of GM2.
4. An antigenic conjugate according to claim 1 wherein the protein
carrier is human serum albumin.
5. A vaccine, which when parenterally injected into a mammal, will
raise the anti-GM2 titer in the blood of said human, said vaccine
consisting essentially of an antigenic conjugate selected from the
antigenic conjugate according to claim 1, claim 2, claim 3 or claim
4, said antigenic conjugate being dissolved in a non-toxic aqueous
solution.
6. A vaccine according to claim 5 wherein the non-toxic aqueous
solution is saline solution.
7. A vaccine according to claim 6 wherein the antigenic conjugate
is present in an amount of between about one to 10 mg. per ml. of
saline solution.
8. A method for increasing the anti-GM2 titer in mammals which
comprises parenterally injecting into said mammals an anti-GM2
increasing titer effective amount of a composition according to
claims 6 or 7.
9. A method for increasing the anti-GM2 titer in mammals in need of
anti-GM2 therapy which comprises parenterally injecting into said
mammals an anti-GM2 increasing titer effective amount of an
antigenic composition which is immuno-reactive with anti-GM2
antibodies produced by mammalian tumors, said antigenic composition
consisting essentially of pure GM2 and a non-toxic stimulative
immune response adjuvant.
10. A method according to claim 9 wherein the stimulative immune
response adjuvant is liposome.
11. A method according to claim 9 wherein the stimulative immune
response adjuvant is lipid A.
12. A method according to claim 9 wherein the non-toxic stimulative
immune response adjuvant is present in an amount of between about
one to 20 parts by weight based on one part by weight of pure GM2.
Description
BACKGROUND OF THE INVENTION
Gangliosides are complex sphingolipids which are found in highest
concentration in the nervous system. They are composed of an
oligosaccharide chain containing an acidic sugar attached to
ceramide. GM2 is a rare ganglioside which is present, in very low
amounts, in normal brain tissue, whose structure is as follows:
##STR1## wherein GalNAc is N-acetylgalactosamine, Gal is galactose,
Glc is glucose and NeuAc is N-acetylneuraminate.
We have found that this ganglioside, GM2, is present on or in human
tumors of a variety of histological types including melanoma, brain
tumors, lung carcinomas, sarcoma and breast carcinomas. The GM2
ganglioside is present on the surface of tumors and stimulates an
appreciable immune response in mammals.
As reported in the publications identified as references 1 through
15 set forth in Table I, a composition, eventually called OFA-I was
found to induce humoral immune responses in cancer patients.
GM2 was identified by use as being an active antigen present in
OFA-I. In vitro tests have demonstrated that anti-OFA-I antibody is
cytotoxic to tumor cells in the presence of human complement
(References 8 and 9, Table I).
Reference 14 in Table I discloses the production of mono-specific
human anti-OFA-I in vitro by the use of human B-lymphocytes
transformed by Epstein-Barr virus.
Reference 4 in Table I discloses the OFA-I on tumor cells is
immunogenic but the chemical nature of the antigenic determinant of
OFA-I is not known.
Reference 15 in Table I discloses an attempt to determine and
purify the antigenic determinant in OFA-I by concentrating the
culture medium, filtering the concentrate and then extracting with
a mixture of chloroform and methyl alcohol. There was obtained a
soluble portion, an insoluble portion and a precipitate. It was
found that the insoluble portion contained oncofetal antigens (OFA)
and the soluble portion contained tumor associated antigens (TAA).
The soluble and insoluble portions were then subjected into a 5-30%
sucrose density radiant ultra-centrifugation. Peak activity of OFA
was found to be present in the 17% sucrose region and peak activity
of TAA was in the 12.5% sucrose.
The article on page 354 states that even though OFA and TAA can be
separated by chloroform-methyl alcohol extraction, the fraction
containing the OFA was "not absolutely pure". On page 355 the
authors state that the "exact chemical nature of the OFA . . .
defined and isolated . . . is unknown at this time."
All of the publications of which we are aware thought that OFA-I,
as defined by serum antibodies, contained only one antigenic
specific determinant which was present on tumor cells and was
immunogenic. However, it was recently discovered by us of the
monospecific antibodies produced in vitro by human B-limphoblastoid
cell lines that there are, in fact, at least two specific antigenic
determinants in OFA-I, GM2 and another ganglioside which is also
immunogenic and present on human tumor cells.
In addition to these two antigens, OFA-I, even when semi-purified
as set forth in reference 15, contains a number of antigenic
compounds (e.g. glycolipids and phospholipids) which makes OFA-I
unsuitable for use as a diagnostic or therapeutic agent because of
interference of the other antigenic compounds.
In addition, insofar as we are aware, antibodies produced by other
gangliosides, when injected into mammals, adversely affect the
nervous system. This is not the case with anti-GM2, presumably
because GM2 is only present in small amounts on the surface of the
brain cells.
SUMMARY OF THE INVENTION
We have discovered that the ganglioside GM2 is the active antigenic
determinant in OFA-I which is immunogenic and which is present on
tumor cells of the histologic type mentioned above.
GM2 cannot be obtained from natural sources since it is present in
extremely small amounts in normal tissues and, insofar as we are
aware, other gangliosides would not be removed. Accordingly, we
have invented a method for producing GM2, which is relatively
simple and inexpensive and renders available large quantities of
GM2 for therapeutic and diagnostic uses. The starting material for
producing GM2 is an abundant ganglioside GM1 which has the
structural formula: ##STR2##
Although OFA-I has been used experimentally in stage II melanoma
patients and it did raise the anti-OFA-I titer, the possibility of
side effects and interference with other antigenic determinants in
OFA-I, makes it impossible to use OFA-I for such purposes, other
than experimental. Accordingly, if GM2 having no other antigens is
conjugated with a non-toxic protein carrier, such as albumin
(preferably serum albumin) or by admixing GM2 with one or more
adjuvants which stimulate immune response (hereinafter stimulative
immune response adjuvants) such as liposomes (hereinafter such
mixtures will sometimes be referred to as antigenic compositions)
it would be an excellent vaccine which will raise the anti-GM2
titer in mammals, e.g. humans. In this regard it is noted that it
is the oligosaccharide portion of GM2 which is antigenic and that
prior to coupling GM2 to the non-toxic protein carrier either the
entire ceramide portion of GM2 may be removed (in which event the
oligosaccharide of GM2 is coupled through the glucose moiety) or
the fatty acid of the ceramide may be removed leaving sphingosine
(in which event the oligosaccharide of GM2 is coupled through the
amine group of the sphingosine moiety).
It is known that patients which have high anti-OFA-I titer have
high survival rates after surgery. Accordingly, the antigenic GM2
conjugate or the GM2-liposome antigenic composition may be used as
a vaccine to immunize cancer patients having histological type
tumors bearing GM2 or as a preventive vaccine to treat
non-cancerous persons in order to prime the immune system to react
against GM2 bearing tumor cells which may appear. Such vaccine may
be administered parenterally, e.g. intradermally or
intralymphatically.
The pure GM2, the conjugated GM2 or the GM2 antigenic composition
are all useful as diagnostic agents. For example, heretofore skin
tests have been used to monitor the effectiveness of immunotherapy
in cancer patients. This type of reaction is the most desirable for
combatting tumor cells. However, heretofore it has not been
possible to test for cell-mediated immunity directed against a
specific antigen. The purified GM2, the conjugated GM2 or the GM2
antigenic composition all may be used for conducting such type of
skin tests, and are particularly useful to test the effectiveness
of the immunization using the GM2 vaccine as described above.
Another diagnostic use for the pure GM2 (i.e. GM2 having no other
antigenic determinants) is to test for circulating (humoral)
antibodies. As has been stated heretofore, it is known that
patients having the histological type tumors will have circulating
anti-GM2. Thus, the pure GM2 may be utilized in an immunoassay
method for determining the amoiunt of anti-GM2 in the serum of
patients. The immunoassay method will include determining the
amount of anti-GM2 present in a liquid sample (e.g. serum) by
mixing with the liquid sample containing the unknown amount of
anti-GM2 an amount of pure GM2, said amount being either more than
the amount of anti-GM2 or less than the amount of anti-GM2 and
allowing said liquid sample containing anti-GM2 and pure GM2 to
incubate until reaction between said anti-GM2 and pure GM2 and then
measuring the amount of one of the following: (1) the amount of
unreacted pure GM2, (2) the amount of unreacted anti-GM2, or (3)
the amount of the reaction product between anti-GM2 and GM2.
Although the quantitative amount of said products may be measured
in any of the numerous methods known to the art, it is preferred if
such measurement is done by utilizing a marker which is easy to
measure quantitatively because of its activity (e.g. the marker may
be an enzyme; a fluorescent molecule which emits light upon
excitation by an appropriate light source; a chemiluminescent
molecule which will emit light after a chemical reaction such as
oxidation; a radioactive molecule; etc.) is linked or bound to
either the antibody or the antigen or the antibody-antigen
conjugate.
One of the simplest methods is to add serum, containing anti-GM2,
to a plastic well which is coated with a small amount of pure GM2.
The antibodies bind to the GM2 in the plastic well and the serum is
then washed out of the well. The antibodies bound to the antigen
are then quantitatively measured by reacting the bound antibody
with a marker such as Protein A or anti-human immunoglobulins
labelled with a radioisotope or conjugated to enzymes (alkaline
phosphatase).
Another diagnostic method of the present invention to determine
cancer is to utilize anti-GM2. We have determined that GM2 is shed
from GM2 bearing tumor cells in tissue culture and GM2 bearing
tumor cells in patients shed GM2 in the serum. Therefore, using the
same method as described above, the amount of GM2 in the serum may
be measured by utilizing anti-GM2.
Another diagnostic test, which this invention is ideally suited, is
tissue typing. By this we mean testing a tumor biopsy for the
presence of GM2. This type of test is important for a number of
reasons, one of the most important being to determine the
effectiveness of immune therapy since tumors devoid of GM2 will be
unaffected by an immune response directed against it. Moreover,
utilizing such tissue typing will enable one to determine whether
such tumor is cancerous by determining whether such tumor has GM2
antigens thereon. Thus, the tissue will be contacted with anti-GM2
and then it will be determined whether the anti-GM2 has reacted
with the tumor cells by measuring the amount of anti-GM2 bound by
direct or indirect methods.
One specific indirect method for accomplishing tissue typing is to
mix anti-GM2 with the tissue homogenate to be tested for GM2 and
the mixture applied to a plastic well coated with GM2. If no GM2 is
present in the tissue samples the antibodies will bind to the well
and after the remaining samples are washed away they can be
detected as discussed above. If GM2 is present in the tissue sample
the antibodies will be bound to the tissue antigen and therefore be
unable to bind to the pure GM2 coating of the plastic well. Thus,
when the sample is washed away, no antibodies will be detected.
Some specific direct methods of determining the amount of GM2
present in tumors would be to determine the binding of anti-GM2
labeled with a marker to frozen tissue sections or to tumor cells
in a tissue homogenate. For frozen tissue, anti-GM2 would be
overlayed on tissue sections and after incubation, unreacted
anti-GM2 would be washed away. The amount, if any, of bound
anti-body would then be determined by measuring the amount of
marker, e.g. by a fluorescent or a light microscope.
For tissue homogenate, anti-GM2 would be added and the mixture
incubated. The tumor cells would be collected by centrifugation and
anti-body bound to the cells would be quantitated by measuring the
amount of the marker present on the cells or the amount of marker
depleted from the supernatant .
DESCRIPTION OF THE PREFERRED EMBODIMENTS
A. Method of making GM2
The general method for the production of the antigenic ganglioside
GM2, which is free of other antigenic determinants (hereinafter
pure GM2) has been set forth above. Such method's starting point is
GM1 which is relatively easy and inexpensive to obtain. The GM1 is
first dissolved in an aqueous solution.
The terminal galactose residue of GM1 is removed, to form GM2, by
using an enzyme effective amount of beta-galactosidase (preferably
lysomal beta-galactosidase). The thus formed GM2 may be separated
from the solution by any of a number of methods including
chromatography on silica gel.
We have found that the reaction is much more efficient if it is
conducted in the presence of a surfactant. We have also found that
a very effective surfactant is a metal (preferably an alkali metal)
salt of taurocholic acid. The surfactant is preferably present in
an amount of between about 3 to 10 parts by weight, based on one
part by weight of the starting ganglioside.
In the preferred method the beta-galactosidase used is bovine
testes beta-galactosidase prepared as described in Reference 16,
Table I.
A reaction solution (1.0 ml) is prepared containing 1 mg. of GM1
and 1 mg. of delipidized bovine serum albumin in 0.01 solar
potassium acetate (pH 5.0), 1 wt. % sodium taurocholate and 0.4
units of bovine testes beta-galactosidase. The solution is
incubated at 37.degree. C. The reaction is greater than 95% in 29
hours. The reaction mixture, after 29 hours, is lyophilized and the
gangliosides, GM1 and GM2, are desalted on a column containing 1.0
gram Sephadex G-25 and eluted.
The eluate is purified by preparative thin layer chromatography in
chlorofrom:methanol:water (1.0:2.0:1.4, vol.). Pure GM2 is eluted
from the silica by extraction into chloroform:methanol (1:2, vol.)
overnight at room temperature (25.degree. C.).
B. GM2 Vaccine
As noted hereinbefore the GM2 vaccine may be of two types: (1) GM2
conjugated to a non-toxic protein carrier and (2) an antigenic
composition consisting essentially of pure GM2 and a stimulative
immune response adjuvent such as liposome or methylated human serum
albumin.
The pure GM2 is conjugated to an appropriate non-toxic carrier such
as human serum albumin using the oligosaccharide of GM2 which is
released by ozonolysis. One mg. of GM2 is dissolved in 1.5 ml. of
absolute methanol. Ozone is bubbled through the solution at room
temperature. The methanol is then evaporated off and after the
methanol is removed, one ml. of 0.1 molar sodium hydroxide is
added. After 16 hours at room temperature, the sample is
neutralized by passage through a column of Dowex 50W-X8 resin
(H.sup.- form). The eluate is lyophilyzed, the residue is dissolved
in 0.002 molar pyridine acetate buffer, pH 5.4, and applied to a
column of DEAE-cellulose equilibrated with the same buffer. The
oligosaccharide is eluted with 1.0 molar pyridine acetate, pH 5.4,
and the eluate lyophilyzed. This yields about 0.5 mg. of the
oligosaccharide portion of GM2.
The oligosaccharide is reacted with beta(p-aminophenethyl)ethyl
amine by mixing the two together at room temperature to form a
Schiff's base conjugate. Sodium borohydride in ethanol is then
added to the conjugate to reduce the conjugate, yielding an
irreversible bond.
After destruction of excess sodium borohydride by lowering the pH
to 5.0 with glacial acetic acid, the ethanol is removed under
vacuum and the resulting conjugate is separated from the free amine
by chromatography on a column of Sephadex G-10.
The now-purified conjugate is linked with thiophosgene and after
removal of the excess thiophosgene by extraction with chloroform,
the isothiocyanate derivative is added to an equal volume of 0.3
molar sodium chloride in 0.1 molar sodium carbonate (pH 9.5)
containing human serum albumin. The mixture is incubated about 18
hours at room temperature and the GM2 oligosaccharide-albumin
conjugate is purified by chromatography on a column of Sephadex
G-75.
In order to use the conjugate as a vaccine for humans it should be
dissolved in an aqueous solution which is non-toxic (e.g. about one
to 10 mg. per ml. of saline). The solution is parenterally injected
(about 5 cc.) every two weeks until high antibody titer appears in
the blood.
In order to use the conjugate as a diagnostic agent for skin tests,
e.g. to determine the effectiveness of the vaccine in cell mediated
delayed hypersensitive, the vaccine solution is injected
intradermally (about 0.5 ml. per site). After about two days the
patient is examined to see if red spots appear at the injected
sites, which indicate the presence of cell mediated immune
response.
The GM2 antigenic composition may be made by mixing pure GM2 with
liposome consisting of dimyristoylphosphatidylcholine (DMPC),
cholesterol and dicetyl phosphate in molar ratios of 2:1.5:0.22 in
a ratio of 0.15 mg. GM2 to one mg. of DMPC. The resulting mixture
is a vaccine which is used in the same way as the protein
conjugate.
The liposome-GM2 antigenic composition may, in general, consist of
one part by weight of GM2 and from about one to 20 parts by weight
of liposome or any other adjuvent known to stimulate immune
response.
Another adjuvant very useful in the present invention is non-toxic
lipid A which may be used per se with GM2 or, preferably, with the
liposome-GM2 antigenic composition. For example, 5 weight parts of
lipid A may be mixed with the liposome composition to produce an
excellent vaccine for therapy of cancer patients or in prevention
of cancer.
C. Diagnostic Tests
Pure GM2 is adsorbed on the walls of polystyrene microtiter plates
by contacting with an aqueous phosphate buffered saline solution of
GM2 (about 1.5 mg. per ml.). The thus coated microtiter plates are
then contacted with 0.05 ml. of serum from a human patient and
allowed to incubate at room temperature for about twelve hours. The
serum is washed off the plates with phosphate buffered saline
solution and the washed microplates are then overlayed with a
solution containing a marker for anti-GM2. The plates are then
incubated for an hour at 25.degree. C. The overlay is removed by
washing any marker remaining on the plate measured by an
approximate instrument which will show the presence or absence of
anti-GM2 in the serum.
TABLE I
References
1. Irie, R. F. and Morton, D. L.: A new membrane antigen on human
cultured cells. Proc. Amer. Assoc. Cancer Res. 16:171, 1975.
2. Irie, R. F. Irie, K. and Morton, D. L.: A membrane antigen
common to human cancer and fetal brain tissues. Cancer Res.
36:3510-3517, 1976.
3. Irie, K., Irie, R. F. and Morton, D. L.: Humoral immune response
of patients with malignant melanomas: Melanoma associated membrane
antigens demonstrable by indirect membrane immunofluorsecence.
Cancer Immunol. Immunother. 6:33-39, 1979.
4. Irie, R. F.: Oncofetal antigen (OFA-I): A human tumor-associated
fetal antigen immunogenic in man. In: Serclogic Analysis of Human
Cancer Antigens (S. Rosenberg, ed.) Academic Press, New York, 1980,
pp. 493-513.
5. Rees, W. V., Irie, R. F. and Morton, D. L.: Oncofetal antigen
(OFA-I): Distribution in human tumors. J. Natl. Cancer Inst.
67:557-562, 1981.
6. Irie, R. F., Giuliano, A. E. and Morton, D. L.: Oncofetal
antigen (OFA): A tumor associated fetal antigen immunogenic in man.
J. Natl. Cancer Inst. 63:367-373, 1979.
7. Sidell, N., Irie, R. F. and Morton, D. L.: Immune cytolysis of
human malignant melanoma by antibody to oncofetal antigen-I
(OFA-I). II. Antibody-dependent cell-mediated cytotoxicity. Cancer
Immunol. Immunother. 9:49-54, 1980.
8. Sidell, N., Irie, R. F. and Morton, D. L.: Oncofetal antigen: A
target for immune cytolysis of human cancer. Brit. J. Cancer
40:950-953, 1979.
9. Sidell, N., Irie, R. F. and Morton, D. L.: Immune cytolysis of
human malignant melanoma by antibody to oncofetal antigen-I
(OFA-I). I. Complement dependent cytotoxicity. Cancer Immunol.
Immunother. 8:211-214, 1980.
10. Jones, P. C., Sidell, N., and Irie, R. F.: Embryonic antigens
in tumor cytolysis (editorial). Cancer Immunol. Immunother.
8:211-214, 1980.
11. Jones, P. C., Sze, L. L., Morton, D. L. and Irie, R. F.:
Prolonged survival for melanoma patients with elevated IfM antibody
to oncofetal antigen (OFA-I). J. Natl. Cancer Inst. 66:249-254,
1980.
12. Irie, R. F., Jones, P. C. and Morton, D. L.: Prolonged survival
of melanoma patients with high titers of antibody to oncofetal
antigen (OFA-I). Proc. Fourth Int. Conf. Immunol. 10.5.35,
1980.
13. Ahn, S. S., Irie, R. F., Weisenburger, T. H., Jones, P. C.,
Juillard, G. J. F., Denise, J. R. and Morton, D. L.: Humoral immune
response to intralymphatic immunotherapy for metastatic melanoma:
Correlation with clinical response. (In press).
14. Irie, R. F., Jones, P. C., and Morton, D. L.: In vitro
production of human antibody to a tumor-associated fetal antigen.
Brit. J. Cancer 44:262-266, 1981.
15. Gupta, R. K., Irie, R. F., Chee, D. O., Kern, D. H., and
Morton, D. L.: Demonstration of two distinct antigens in spent
tissue culture medium of human malignant melonoma cell line. J.
Nat, Cancer Inst. 63:359-366, 1980.
16. Distler, J. J. and Jourdian, G. W.: The purification and
properties of .beta.-galactosidase from bovine testes. J. Biol.
Chem. 248: 6772-6780, 1973.
* * * * *